Magnetically responsive composition

ABSTRACT

A magnetically controllable, or guided, ferrocarbon particle composition and methods of use and production are disclosed. The composition may optionally carry biologically active substances that have been adsorbed onto the particle. The composition comprises composite, volume-compounded particles of 0.1 to 5.0 μm in size, and preferably between 0.5 and 5.0 μm, containing 1.0 to 95.0% by mass of carbon, and preferably from about 20% to about 60%. The particles may be produced by mechanical milling of a mixture of iron and carbon powders. The obtained particles are optionally placed in a solution of a biologically active substance to adsorb the substance onto the particles. The composition is generally administered in suspension.

RELATED APPLICATION

[0001] This Application is a continuation-in-part of now pending U.S.application Ser. No. 09/003,286, which is a continuation-in-part of U.S.patent application Ser. No. 08/480,195, filed Jun. 7, 1995 (now U.S.Pat. No. 5,705,195), which is a continuation of U.S. application Ser.No. 08/188,062, filed Jan. 26, 1994 (now U.S. Pat. No. 5,549,915), whichis a continuation-in-part of U.S. patent application Ser. No.08/011,363, now abandoned

FIELD OF INVENTION

[0002] This invention relates to compositions and methods for deliveryof biocompatible particles to a selected location in a body, and, moreparticularly, relates to particles capable of carrying biologicallyactive compounds, which provide for targeted magnetic transport of theparticles and the maintenance of them in a predetermined place forlocalized diagnostic or therapeutic treatment of disease.

BACKGROUND OF THE INVENTION

[0003] Metallic carrier compositions used in the treatment of variousdisorders have been heretofore suggested and/or utilized (see, forexample, U.S. Pat. Nos. 4,849,209 and 4,106,488), and have included suchcompositions that are guided or controlled in a body in response toexternal application of a magnetic field (see, for example, U.S. Pat.Nos. 4,501,726, 4,652,257 and 4,690,130). Such compositions have notalways proven practical and/or entirely effective. For example, suchcompositions may lack adequate capacity for carriage of the desiredbiologically active agent to the treatment site, have less thandesirable magnetic susceptibility and/or be difficult to manufacture,store and/or use.

[0004] One such known composition, deliverable by way of intravascularinjection, includes microspheres made up of a ferromagnetic componentcovered with a biocompatible polymer (albumin, gelatin, andpolysaccharides) which also contains a drug (Driscol C. F. et al. Prog.Am. Assoc. Cancer Res., 1980, p. 261).

[0005] It is possible to produce albumen microspheres up to 3.0 μm insize containing a magnetic material (magnetite Fe₃O₄) and theanti-tumoral antibiotic doxorubicin (Widder K. et al. J. Pharm. Sci.,68:79-82 1979). Such microspheres are produced through thermal and/orchemical denaturation of albumin in an emulsion (water in oil), with theinput phase containing a magnetite suspension in a medicinal solution.Similar technique has been used to produce magnetically controlled, orguided, microcapsules covered with ethylcellulose containing theantibiotic mitomycin-C (Fujimoto S. et al., Cancer, 56: 2404-2410,1985).

[0006] Another method is to produce magnetically controlled liposomes200 nm to 800 nm in size carrying preparations that can dissolveatherosclerotic formations. This method is based on the ability ofphospholipids to create closed membrane structures in the presence ofwater (Gregoriadis G., Ryman B. E., Biochem. J., 124:58, 1971).

[0007] The above compositions require extremely high flux densitymagnetic fields for their control, and are somewhat difficult to produceconsistently, sterilize, and store on an industrial scale withoutchanging their designated properties.

[0008] To overcome these shortcomings, a method for producingmagnetically controlled dispersion has been suggested (See EuropeanPatent Office Publication No. 0 451 299 A1, by Kholodov L. E., VolkonskyV. A., Kolesnik N. F. et al.), using ferrocarbon particles as aferromagnetic material. The ferrocarbon particles are produced byheating iron powder made up of particles 100 μm to 500 μm in size attemperatures of 800° C. to 1200° C. in an oxygen-containing atmosphere.The mixture is subsequently treated by carbon monoxide at 400° C. to700° C. until carbon particles in an amount of about 10% to 90% by massbegin emerging on the surface. A biologically active substance is thenadsorbed on the particles

[0009] This method of manufacturing ferrocarbon particles is rathercomplicated and requires a considerable amount of energy. Because theferromagnetic component is oxidized due to the synthesis of ferrocarbonparticles at a high temperature in an oxygen containing atmosphere,magnetic susceptibility of the dispersion obtained is decreased by aboutone-half on the average, as compared with metallic iron. The typicalupper limit of adsorption of a biologically active substance on suchparticles is about 2.0% to 2.5% of the mass of a ferromagnetic particle.

[0010] The magnetically controlled particle produced by the above methodhas a spherical ferromagnetic component with a thread-like carbon chainextending from it and is generally about 2.0 μm in size. The structureis believed to predetermine the relatively low adsorption capacity ofthe composites and also leads to breaking of the fragile thread-likechains of carbon from the ferromagnetic component during storage andtransportation.

[0011] Thus, there remains a need for an effective biocompatiblecomposition which is capable of being transported magnetically, andwhich is relatively easy to manufacture, store and use.

SUMMARY OF THE INVENTION

[0012] This invention provides a magnetically responsive compositionwhich may carry biologically active substances, or which may be usedalone. Generally, any soluted substance can be carried, many of whichhave been heretofore suggested. For example, without limitation,alkylating agents, antimetabolites, antifungals, anti-inflammatory,antitumor, and chemotherapy agents, and suitable combinations thereofcan be adsorbed on the particles. Other therapeutic agents and drugs,such as systemic toxicity inhibitors, antibiotics and hydrocortisone, orthe like, can also be carried and administered in vivo by use of themagnetically controlled carrier particles of the invention. Methods ofproduction and use thereof are also provided.

[0013] The aim of this invention is to improve some parameters ofmagnetically controlled compositions used for the targeted transport ofbiocompatible particles, including increasing relative adsorptioncapacity, increasing magnetic susceptibility, intensifying diagnosticand therapeutic effect and ease of use, as well as simplifying thetechnology of manufacturing the magnetically controlled composition andensuring its guaranteed long storage without changing its desiredcharacteristics.

[0014] This is achieved by using suitable composite, volume compoundedferrocarbon particles as a magnetically susceptible material for amagnetically controlled composition. These particles have a majordimension (i.e., largest diameter) of about 0.2 μm to about 5.0 μm (andpreferably from 0.5 μm to 5.0 μm) and contain from about 1.0% to about95.0% (by mass) of carbon, with the carbon strongly connected to iron.The particles are obtained by jointly deforming (i.e., milling) amixture of iron and carbon powders. In some cases the finished particlesinclude trace amounts of the compound cementite (Fe₃C).

[0015] The composition utilized for localized in vivo treatment ofdisease includes particles of about 0.5 μm and 5 μm in major dimension,each particle including carbon and iron and, optionally, a biologicallyactive substance selected for its efficacy in diagnosing or treating thedisease adsorbed on the particles.

[0016] The method of producing the composition includes the step ofjointly deforming a mechanical mixture of iron and carbon powders for atime sufficient to bind the powders into a composite of iron:carbonparticles having an average major dimension of less than 5 μm in size,and with a substantial portion of the particles including about 1.0% to95.0% by mass of carbon distributed throughout the volume of each of theparticles. The particles are preferably separated to select particleshaving a major dimension of from about 0.5 μm to about 5.0 μpm, afterwhich up to 20% by mass of the particles of a biologically activesubstance can be adsorbed onto the selected particles.

[0017] The methods of use include methods for localized in vivodiagnosis or treatment of disease comprising providing a magneticallyresponsive ferrocarbon carrier (such as the carrier of this invention)having adsorbed thereon a biologically active substance selected for itsefficacy in diagnosing or treating the disease, and injecting thecarrier into the body of a patient. For example, the carrier is injectedby inserting delivery means into an artery to within a short distancefrom a body site to be treated and at a branch or branches (preferablythe most immediate) to a network of arteries carrying blood to the site.The carrier is injected through the delivery means into the bloodvessel. Just prior to injection, a magnetic field is establishedexterior to the body and adjacent to the site of sufficient fieldstrength to guide a substantial quantity of the injected carrier to, andretain the substantial quantity of the carrier at, the site. Preferably,the magnetic field is of sufficient strength to draw the carrier intothe soft tissue at the site adjacent to the network of vessels, thusavoiding substantial embolization of any of the larger vessels by thecarrier particles.

[0018] It is therefore an object of this invention to provide animproved magnetically responsive composition for optionally carryingbiologically active substances and methods of production and usethereof.

[0019] It is another object of this invention to provide a magneticallyresponsive carrier for biologically active substances which has improvedmagnetic responsiveness, yet is durable during storage and use, andincludes up to about 20% by mass of a biologically active substanceadsorbed thereon.

[0020] It is another object of this invention to provide a magneticallyresponsive composition comprising particles having a major dimension offrom about 0.5 μm to about 5.0 μm, each iron:carbon composite particleincluding about 1.0% to about 95.0% by mass of carbon distributedthroughout the volume of the particle.

[0021] It is still another object of this invention to provide acomposition utilized for localized in vivo diagnosis or treatment ofdisease including a carrier with composite iron:carbon particles fromabout 0.5 μm to about 5.0 μm in size, each composite iron:carbonparticle including carbon and iron with the carbon distributedthroughout the volume of the particle, and an optional biologicallyactive substance selected for its efficacy in diagnosing or treating thedisease which is adsorbed on the particles.

[0022] It is yet another object of this invention to provide a method ofproducing a magnetically responsive carrier composition includingcomposite iron:carbon particles including carbon and iron with thecarbon distributed throughout the volume of each of the particles.

[0023] It is yet another object of this invention to provide liquid anddry kits for administering a composition utilized for localized in vivodiagnosis or treatment of disease including a ferrocarbon particle withan optional biologically active substance adsorbed thereon that has beenselected for its efficacy in diagnosing or treating the disease.

[0024] It is a further object of this invention to provide methods ofsterilization of the components of the kits supplied for administering acomposition utilized for localized in vivo diagnosis or treatment ofdisease including a ferrocarbon particle with an optional biologicallyactive substance adsorbed thereon that has been selected for itsefficacy in diagnosing or treating the disease.

[0025] With these and other objects in view, which will become apparentto one skilled in the art from the following description, this inventionresides in the novel construction, combination, arrangement of parts andmethods substantially as hereinafter described, and more particularlydefined by the appended claims, it being understood that changes in theprecise embodiment of the herein disclosed invention are meant to beincluded as they come within the scope of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 is a magnified photograph (12000×) of composite particlesof the carrier composition of this invention.

[0027]FIG. 2A is a magnified photograph (30,000×) of a particle of thecarrier composition of this invention.

[0028]FIG. 2B is a sectional illustration of the particle of FIG. 2A.

[0029]FIGS. 3A through 3H are illustrations of a tumor during periods oftreatment utilizing drugs adsorbed on the carrier composition anddelivered to, and maintained at, the tumor site utilizing one method ofthis invention.

[0030]FIG. 4 is a diagram illustrating one example of application andmagnetic targeting of the carrier composition.

[0031]FIG. 5 is a diagram illustrating the carrier composition (having adrug adsorbed thereon) at a pathological structure.

[0032]FIG. 6 is a graph showing Langmuir adsorption plots for PACbinding to (—O—) carrier particles with an iron:carbon ratio of 70:30Type E carbon and (-¤-) Type E carbon alone. Data were fit by simpleunweighted linear regression.

DESCRIPTION OF THE INVENTION

[0033] The magnetically controllable, or guided, carrier composition ofthis invention includes composite, volume-compounded ferrocarbonparticles of about 0.1 μm to about 5.0 μm in average major dimension,and preferably between about 0.5 μm and about 5.0 μm, containing about1.0% to about 95.0% by mass of carbon, for example, between about 10%and 60%. About 20% to about 40% is the preferred range of carbon havingbeen found to exhibit characteristics useful in many applications.

[0034] The particles are produced by mechanically milling a mixture ofiron and carbon powders, without application of external heat. Thecomposite iron:carbon carrier particles so obtained may then be placedin a solution of a biologically active substance to allow adsorption ofthe biologically active substance to the particles. The compositeparticles are separated for desired size and magnetic susceptibilitycharacteristics. Separation of the particles can occur before orsubsequent to exposure to the biologically active substance.

[0035] As shown in FIGS. 1 and 2A, iron:carbon particles 8 manufacturedby the method of this invention are of a generally spherical shape, withthe inclusions of carbon deposits 10 presumably being located throughoutthe whole volume of each particle (both at the surface and the interiorof each particle). The strong connection between the components (iron 12and carbon 10) which is not broken during prolonged storage of themagnetically controlled composition, its transportation, storing,packing and direct use. Chemical binding may take place between the ironand carbon, such as a trace interlayer of cementite (Fe₃C) formed duringthe milling process.

[0036] The iron:carbon particles are also useful as a carrier fordelivering one or more adsorbed biologically active substances tospecific body sites under control of an external magnetic field. As usedherein, the term “biologically active substance” includes substancesuseful for in vivo medical diagnosis and/or treatment.

[0037] Biologically active substances include, but are not limited toantineoplastics, blood products, biological response modifiers,anti-fungals, antibiotics, hormones, vitamins, peptides, enzymes, dyes,anti-allergics, anti-coagulants, circulatory agents, metabolicpotentiators, antituberculars, antivirals, antianginals,anti-inflammatories, antiprotozoans, antirheumatics, narcotics, opiates,diagnostic imaging agents, cardiac glycosides, neuromuscular blockers,sedatives, anesthetics, as well as paramagnetic and radioactiveparticles. Other biologically active substances may include, but are notlimited to monoclonal or other antibodies, natural or synthetic geneticmaterial and prodrugs.

[0038] As used herein, the term “genetic material” refers generally tonucleotides and polynucleotides, including nucleic acids, RNA and DNA ofeither natural or synthetic origin, including recombinant, sense andantisense RNA and DNA. Types of genetic material may include, forexample, genes carried on expression vectors, such as plasmids,phagemids, cosmids, yeast artificial chromosomes, and defective (helper)viruses, antisense nucleic acids, both single and double stranded RNAand DNA and analogs thereof, as well as other proteins or polymers.

[0039] For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay that is detectable for agiven type of instrument. Generally, gamma radiation is required. Stillanother important factor in selecting a radioisotope is that thehalf-life be long enough so that it is still detectable at the time ofmaximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Selection of anappropriate radioisotope would be readily apparent to one having averageskill in the art. Radioisotopes which may be employed include, but arenot limited to ⁹⁹Tc, ¹⁴²Pr, ¹⁶¹Tb, ¹⁸⁶Re, and ¹⁸⁸Re. Additionally,typical examples of other diagnostically useful compounds are metallicions including, but not limited to ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr,and ²⁰¹TI. Furthermore, paramagnetic elements that are particularlyuseful in magnetic resonance imaging and electron spin resonancetechniques include, but are not limited to ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe.

[0040] It is also noted that radioisotopes are also useful in radiationtherapy techniques. Generally, alpha and beta radiation is considereduseful for therapy. Examples of therapeutic compounds include, but arenot limited to ³²P, ¹⁸⁶Re, ¹⁸⁸Re, ¹²³I, ¹²⁵I, ⁹⁰Y, ¹⁶⁶Ho, ¹⁵³Sm, ¹⁴²Pr,¹⁴³Pr,¹⁴⁹Tb,¹⁶¹Tb, ¹¹¹In, ⁷⁷Br, ²¹²Bi, ²¹³Bi, ²²³Ra, ²¹⁰Po, ¹⁹⁵Pt,^(195m)Pt, ²⁵⁵Fm, ¹⁶⁵Dy, ¹⁰⁹Pd, ¹²¹Sn, ¹²⁷Te, and ²¹¹At. Theradioisotope generally exists as a radical within a salt, however sometumors and the thyroid may take up iodine directly. The usefuldiagnostic and therapeutic radioisotopes may be used alone or incombination.

[0041] As a general principle, the amount of any aqueous solublebiologically active substance adsorbed can be increased by increasingthe proportion of carbon in the particles up to a maximum of about 40%by mass of the composite particles without loss of utility of theparticles in the therapeutic treatment regimens described in thisapplication. In many cases it has been observed that an increase in theamount of adsorbed biologically active substance is approximately linearwith the increase in carbon content. However, as carbon contentincreases, the susceptibility, or responsiveness, of composite particles8 to a magnetic field decreases, and thus conditions for their controlin the body worsen (although adsorption capacity increases). Therefore,it is necessary to achieve a balance in the iron:carbon ratio to obtainimproved therapeutic or diagnostic results. To increase the amount ofagent given during a treatment regimen, a larger dose of particles canbe administered to the patient, but the particles cannot be made moremagnetic by increasing the dose. Appropriate ratios may be determined byany person having average skill in the art.

[0042] It has been determined that the useful range of iron:carbon ratiofor particles intended for use in in vivo therapeutic treatments asdescribed in the application is, as a general rule, from about 95:5 toabout 50:50, for example about 80:20 to about 60:40. The maximum amountof the biologically active substance that can be adsorbed in thecomposite iron:carbon carrier particles of any given concentration ofcarbon will also differ depending upon the chemical nature of thebiologically active substance, and, in some cases, the type of carbon(i.e., activated carbon (AC)) used in the composition. For example, ithas been discovered that the optimal iron:carbon ratio for carrierparticles used to deliver adsorbed doxorubicin in in vivo therapeutictreatments is about 75:25.

[0043] However, adsorption of biologically active substances that aresubstantially insoluble in water (i.e., with solubility in water lessthan about 0.1% by weight) requires use of special procedures to adsorba useful amount of a drug on the particles. Applicants have discoveredthat adsorption on the carrier particles of this invention ofbiologically active substances having substantial insolubility in watercan be obtained without the use of surfactants, many of which are toxic,by dissolving the water insoluble biologically active substance in aliquid adsorption medium (e.g., aqueous) that includes excipientsselected to minimize the hydrophobic Van der Waals forces between theparticles and the solution and to prevent agglomeration of the particlesin the medium. For example, if the biologically active substance is ahighly non-polar molecule, such as camptothecin, and the adsorptionmedium is a highly non-polar liquid, such as chloroform-ethanol, thedrug does not preferentially leave the adsorption medium to adsorb tothe carbon. However, in a more polar adsorption medium, adsorption tothe carrier particles is entirely acceptable. For example, binding oftherapeutic levels of paclitaxel, a highly water-insoluble drug, tocarrier particles having an iron:carbon ratio of 70:30 was obtainedusing citrated ethanol as the adsorption medium, even though paclitaxelis substantially water insoluble. In many cases, it is advantageous ifthe liquid adsorption medium includes a biologically compatible andbiodegradable viscosity-increasing agent (e.g., a biologicallycompatible polymer), such as sodium carboxymethyl cellulose, to aid inseparation of the particles in the medium.

[0044] Using the methods of this invention, doxorubicin has beenadsorbed onto carrier particles having iron:carbon ratios from 80:20 to60:40 (Type A activated carbon) in amounts in the range from about 0.0%to about 20% of the mass of the particles on average. Example 5illustrates the formulation of excipients useful for enhancingadsorption of doxorubicin to the carrier particles. Other biologicallyactive agents may also be adsorbed using similar techniques that wouldbe obvious to any person having average skill in the art.

[0045] Because it is convenient to prepare and market the carrierparticles in a dry form, the excipients may be prepared in dry form, andan adsorption-enhancing amount of one or more dry excipients useful forsolubilizing the drug or other biologically active substance when in aliquid solution are packaged together with a unit dose of the carrierparticles. An adsorption-enhancing amount of the dry excipients will bedetermined by one of skill in the art depending upon the chemicalproperties of the biologically active substance as needed to overcomethe chemical forces that cause insolubility of the biologically activesubstance of interest and agglomeration of the particles in aqueoussolution. Most preferably, the package or kit containing both the dryexcipients and dry carrier particles is formulated to be mixed with thecontents of a vial containing a unit dose of the drug and sufficientamount of a biologically compatible aqueous solution, such as saline, asrecommended by the drug manufacturer, to bring the drug to apharmaceutically desirable concentration. Upon mixture of the solutioncontaining the dilute drug with the contents of the kit including thedry components (i.e., the dry carrier particles and dry excipients), thedrug is allowed to adsorb to the carrier particles, forming amagnetically controllable composition containing a therapeutic amount ofthe biologically active substance adsorbed to the carrier particles thatis suitable for in vivo therapeutic or diagnostic use.

[0046] Alternatively, a liquid kit may be employed. Here, the carrierparticles are contained as one unit, for example, in a vial, while theaforementioned excipients are contained in another unit in the form ofan aqueous solution. At the time of administration, the ferrocarbonparticles are mixed with the contents of a vial containing a unit doseof the drug and sufficient amount of a biologically compatible aqueoussolution, such as saline, as recommended by the drug manufacturer, tobring the drug to a pharmaceutically desirable concentration.Subsequently, the resulting particles having the biologically activesubstance adsorbed thereon, are mixed with yet another unit containingthe excipients in aqueous solution. Any suitable sterilization techniquemay be employed. For exarnple, the ferrocarbon particles may besterilized using gamma irradiation and the aqueous solution ofexcipients may be sterilized by autoclave. Use of autoclave undesirablyoxidizes the ferrocarbon particles.

[0047] A diagnostic or therapeutic amount of biologically activesubstance adsorbed to the carrier particles will be determined by oneskilled in the art as that amount necessary to effect diagnosis ortreatment of a particular disease or condition, taking into account avariety of factors such as the patient's weight, age, and generalhealth, the diagnostic or therapeutic properties of the drug, and thenature and severity of the disease.

[0048] A number of considerations are involved in determining the sizeof carrier particles to be used for any specific therapeutic situation.The choice of particle size is determined in part by technologicalconstraints inherent in producing the particles under 0.2 μm in size. Inaddition, for particles less than about 1.0 μm in size, the magneticcontrol in blood flow and the carrying capacity is reduced. Relativelylarge particle sizes can tend to cause undesirable embolization of bloodvessels during injection either mechanically or by facilitating clotformation by physiological mechanisms. The dispersion may coagulate,which makes injections more difficult, and the rate at whichbiologically active substances desorb from the particles in the targetedpathological zones may decrease. The method (such as is described below)of milling together a mixture of iron and carbon powders produces aproduces an approximately spherical form with a granular surface for theparticles, and results in a particle population having an average majordimension of about 0.1 μm to about 5.0 μm.

[0049] Because the iron in the particles described in this invention isnot in the form of an iron oxide, as is the case in certain previouslydisclosed magnetically controlled dispersions, the magneticsusceptibility, or responsiveness, of ferrocarbon particles 8 ismaintained at a high level.

[0050] The iron:carbon particles are characterized by a well-developedsubstructure (see FIG. 2B), having a connected network of iron forming anetwork of voids with carbon deposits 10 captured therein. Thecharacteristic substructure of the particles formed during the processof joint deformation of the mechanical mixture of iron and carbonpowders, also increases the magnetic susceptibility of iron inclusionsin ferrocarbon particles 8 as compared with iron particles having othertypes of substructure. For example, the composite ferrocarbon particlesproduced by the herein suggested method have greater magneticsusceptibility than the particles disclosed in European Patent OfficePublication No. 0 451 299 A1, although the ferromagnetic content in bothtypes of particles is about the same. This high magnetic responsivenessof ferrocarbon particles 8 makes it possible, in some cases, to utilizemagnetic fields lower than about 250 gauss to position the particles atthe desired anatomical site.

[0051] Because of the large surface of carbon deposits 10 in particles8, the adsorbed biologically active substance comprises up to about20.0% by mass of particles 8; or, in different terms, up to about 200 mgof adsorbed biologically active substance per gram of particles 8.Therefore, in use, much less of the carrier is injected to achieve agiven dose of the biologically active substance or, alternatively, ahigher dosage of the biologically active substance per injection isobtained than is the case with some previously known carriers.

[0052] The following describes a method for producing small quantitiesof the ferrocarbon composition of this invention, it being understoodthat other means and mechanisms besides milling could be conceived offor jointly deforming iron and carbon powders, which comprise theessential starting elements for production of the carrier. The procedureutilized exerts mechanical pressure on a mixture of carbon and ironparticles to deform the iron particles and develop a substantialsubstructure, which captures the carbon. The formation of theferrocarbon particles is accomplished without the addition of heat inthe process (although the mixture heats up during the mechanicaldeformation step), and is conducted in the presence of a liquid, forexample ethanol, to inhibit oxidation of the iron and to assure that theparticles produced are clean (sterile). The liquid may also serve as alubricant during the milling of the iron and carbon powder, and mayreduce compacting of carbon during processing. As a result, the densityof the carbon deposits in the composition is maintained so as tomaximize adsorption capacity of the particles.

[0053] For example, to produce particles having an average of about75:25 iron:carbon ratio by mass, one part of substantially pure ironparticles having average diameters from 0.1 μm to 5 μm in size are mixedwith about 0.1 to 1.0 parts by weight of substantially pure carbongranules (typically about 0.1 μm to 5.0 μm in diameter). The ironparticles and carbon granules are mixed vigorously to achieve gooddistribution throughout the volume. Preferably the carbon granules areactivated carbon. Each biologically active substance should be evaluatedindividually with the various types of carbon in order to determine theoptimum reversible activated carbon binding. Factors such as pH,temperature, particulate size, salts solution viscosity and otherpotentially competing chemicals in solution can influence adsorptioncapacity, rate, and desorption parameters. Activated carbon types whichare useful include, but are not limited to A, B, E, K and KB andchemically modified versions thereof.

[0054] The mixture is put into a standard laboratory planetary ball, orattrition mill of the type used in powder metallurgy. For example, themill can have 6 mm diameter balls. An appropriate amount of a liquid,for example ethanol, is added for lubrication. The mixture is milled forbetween 1 and 12 hours, or for the time necessary to produce theparticles heretofore described. Depending on the mill used, the speed ofthe mill may be anywhere in the range from about 120 rpm to about 1000rpm (typically about 350 rpm), the process not being overly sensitive tothe speed of the mill.

[0055] After joint deformation of the iron:carbon mixture, the particlesare removed from the mill and separated from the grinding balls, forexample, by a strainer. The particles may be resuspended in ethanol andhomogenized to separate the particles from each other. The ethanol isremoved, for example, by rotary evaporation, followed by vacuum drying.Any suitable drying technique may be employed. Particles should behandled so as to protect against oxidation of the iron, for example, ina nitrogen environment.

[0056] After drying, the particles should be collected according toappropriate size. For example, the particles may be passed through a 20μm sieve and collected in an air cyclone to remove particles larger than20 μm. The cyclone only collects particles of a certain size anddensity, providing a method for removing fines and loose carbon. Thesieved particles may be packaged under nitrogen and stored at roomtemperature.

[0057] Particles may be subaliquoted into dosage units, for example,between 50 and 500 μg per dose, and may be further overlayed withnitrogen, for example. Dosage units may be sealed, for example, withbutyl rubber stoppers and aluminum crimps. Dosage units may then besterilized by appropriate sterilization techniques, for example, gammairradiation between 2.5 and 3.5 Mrads.

[0058] When ready for use, or before packaging if the carrier is to beprepared with a preselected biologically active substance alreadyadsorbed thereon, about 50 mg to 150 mg (about 75 mg to about 100 mg ispreferred to be absolutely assured of maximum adsorption) of thebiologically active substance in solution is added to I gram of thecarrier. When ready for application to a patient, the combination isplaced into suspension (for example, in 5 to 10 ml) of a biologicallycompatible liquid such as water or saline utilizing normal procedures.

[0059] Experimental evidence shows increased therapeutic efficacy on atumor growth with the use of the magnetically controlled carriercomposition of this invention with an anti-tumor preparation incomparison with previously known magnetically controlled dispersions.

EXAMPLE 1

[0060] Tests were carried out on male rats of the Wistar Line (bred atStolbovaya Station of the USSR Academy of Medical Sciences). The ratswere infused with carcinosarcoma Walker 256 under the tail skin. Whenthe tumorous volume averaged 986±98 mm³ the animals were divided into 4groups, 10 rats in each. The first group (group I) was a control group,and groups II through IV were experimental groups.

[0061] The animals in group II were given intravenous injections of awater solution of rubomicine in the amount of 2 mg/kg of body weightduring 5 days (the model of traditional use of such anti-cancerouspreparations in clinics). The rats in group III were injected with asuspension of ferrocarbon dispersion produced by the previously knownmethod described in European Patent Office Publication No. 0 451 299 A1.The particles comprised iron/carbon in a volume percent ratio of 60:40.The dosage of ferrocarbon particles was 160 mg/kg of body weight, andthe dosage of adsorbed rubomicine thereon was 3.2 mg/g of particles.This suspension was injected into the tail vein after placing on thesurface of the tumor a permanent magnet with a magnetic field intensityof 6000 oersteds. Localization of the suspension in the tumorous growthzone under control of the externally placed magnetic field was monitoredby x-ray pictures.

[0062] Using the same techniques for injection and magneticlocalization, including placement of a permanent magnet with a magneticfield of 600 oersteds on the surface of the tumor and monitoring. Theanimals from group IV were given a one-time intravenous injection of themagnetically controlled dispersion produced in accord with the methodsof this invention localization of the particles was observed by x-ray.The dosage was 160 mg of carrier particles per/kg of carrier particlesof body weight. The combination of iron:carbon in individual particlesof the dispersion was in percent ratio of 60:40, which was similar tothe ratio in the dispersion produced by the previously known method usedin experimental group III.

[0063] Due to the improved adsorptive capability of particles 8, thedose of rubomicine adsorbed on the magnetically controlled carrierparticles of this invention was 9.96 mg of rubomicine per/g ofparticles, which was 3.1 times more than the rubomicine adsorbed by thepreviously known carrier particles in the experiment with the rats ofgroup III. This result was achieved solely due to the relative specificadsorption capacities of the given carrier particles.

[0064] Observation of the animals gave the following results. The lifespan of animals in control group I averaged 21±1.5 days. In group II, asa result of prescribed intravenous injections of the water solution ofrubomicine, to model the traditional use of anti-tumor drugs in theclinic, the life span of the rats following treatment increased by anaverage of 4.5 days (P<0.05). The animals from experimental group IIIlived for an average of 46±4.3 days following treatment, which was 2.2times more (P>0.001) than the life span of the control animals.

[0065] In group IV, 6 rats out of 10 (i.e., 60% of the cases)demonstrated complete dissolution of the tumor, which took place during5 to 7 days after the one-time injection of the suspension of themagnetically controlled composition. Moreover, the remaining 4 rats fromthis group lived an average 57.4±5.9 days after treatment, thusexceeding the life span of the animals from group III by 25.0%. Theiraverage life span post treatment was also 2.7 times longer than that ofthe rats from control group I. The animals from group IV that showedcomplete regression of the tumors did not see any recurrence of tumorousgrowth during 157 days of observation, which is a result consistent withcomplete elimination of the tumors in these rats.

EXAMPLE 2

[0066] Further clinical observation has documented the effectiveness ofthis invention. FIGS. 4 and 5 illustrate use of this invention fortreatment and observation of a 61 year-old woman admitted on Feb. 13,1992, to the Zil Hospital in Moscow, Russia (CIS) and diagnosed withcancer of the left mammary gland T₃N₁M_(l).

[0067] The diagnosis was first made in 1989 when a biopsy was done. InDecember, 1991, focal radiation therapy (10 grey) resulted in the tumorbeing partially reduced. The decision was made to use chemotherapy inthe forms of the intra-arterial selective localization of the carrier ofthis invention with doxorubicin (Adriamycin®) as the biologically activeagent adsorbed on the carrier.

[0068] Before the treatment, the dimensions of the tumor (illustrated inFIGS. 3A and 3B) were 44 mm×33 mm×37 mm (65 mm×45 mm, manual). On Feb.24, 1992, a femoral artery (FIG. 4) was punctured and a vascularcatheter was inserted into the aorta according to the Seldinger methodunder local anaesthesia (0.5% novocaine, 30 ml). Under roentgenologicand contrast control, the catheter was placed at 25 mm distance from thebranch to the left intra-pectoral artery (a. mammaria interna sinisra).A newly prepared suspension of gelatinol with ferrocarbon particles 8having 15 mg doxorubicin (Adriamycin) adsorbed thereon was injectedthrough the catheter. At this time, a magnet having a magnetic fieldintensity of 15,000 oersteds was placed over the tumor for 20 minutes.As a result, the injected suspension was kept localized by the magneticfield in the zone of the tumor for 20 minutes (a time sufficient forfull microembolization of the tumor feeding capillaries). The patient'scondition was satisfactory at the time of therapy.

[0069] By Feb. 28, 1992, the patient's condition had improved. Anultrasonic examination of the left mammary gland showed the dimensionsof the tumor at 42 mm×33 mm×40 mm as shown in FIGS. 3C and 3D. The tumorhad a legible contour. By Mar. 12, 1992, the dimensions of the tumor hadbeen reduced by 66.3% to 32 mm×27 mm×21 mm (FIGS. 3E and 3F). By Apr.14, 1992, the dimension had been reduced by 99.22% to 10 mm×6 mm×7 mm(FIGS. 3G and 3H).

[0070] It is believed that by releasing the carrier immediately upstreamof the tumor (or other pathological) site, rather than penetrating thetumor, equally effective application of the biologically activesubstance occurs while potentially benefiting the patient by limitingspread of disease occasioned by puncture of the tumorous tissue. While alarger magnetic field was utilized in the above example of treatment, ithas been found that the carrier composition of this invention begins toreact in a field as small as 250 oersteds/cm (many prior art carriersneeding a field as large a 500 oersteds/cm before being influenced).

[0071]FIG. 5 illustrates what is believed to occur under magneticcontrol at the treatment site. Under the influence of the appliedmagnetic field, the carrier particles are induced into the capillarynetwork feeding the tumor. The particles are drawn closely adjacent tothe soft tissue of the lumen of the capillaries (or perhaps even intothe soft tissue) thereby reducing or eliminating the potential forembolization of the vessels by the carrier particles. The biologicallyactive substance is released from the carrier particles by a dynamicprocess in which the substance in the carrier is replaced by materialsproduced by the body. For example the necrotic products of the tumoritself, may replace the biologically active substances, becomingadsorbed on the carrier particles such as proteins, glucose, lipids,peptides, or the like. Thus, the biologically active substance isliterally pushed out of the carrier particles.

[0072] Typically, less than about 10% of the biologically activesubstance is replaced by body materials in the blood stream. Therefore,it is believed that the replacing substance must have a higher specificgravity than that of the biologically active substance. A small amountof the particles may not be attracted to the treatment site by themagnetic field or escapes from the treatment site. This fraction mayalso therapeutically active against tumor cells in the blood andelsewhere. In some cases, reduction in metastasis has been observedfollowing treatment according to the method of this invention. Since thecarrier composition is formed of material that is biodegradable or canbe readily metabolized by the body, all carrier particles are excretedor metabolized, perhaps within 30 days of application.

[0073] As may be appreciated, an improved magnetically responsivecarrier for biologically active substances and methods for producing andusing the same are provided by this invention. The carrier particlesexhibit improved responsiveness to magnetic fields, have improved drugadsorptive capacity, and are more durable during storage and use.

EXAMPLE 3

[0074] Recently a series of fluoroscopically-guided organ imagingstudies were conducted in a porcine animal model using radioactivetechnetium (Tc) adsorbed to the carrier particles of the invention asthe imaging agent. In order to evaluate physical chemical properties andinteraction of Tc with carbon (C) and the iron:carbon carrier particles,rhenium (Re) was used as a non-radioactive surrogate for Tc. Re is agroup VIIB element just below Tc in the periodic table. It has twoartificial isotopes, ¹⁸⁶Re and ¹⁸⁸Re, which have half-lives longer thanthat of Tc and emit about the same gamma radiation as shown in Table 1below: TABLE 1 Isotope source t_(½ in hours) Gamma energy (keV) ⁹⁹Tcartificial  7 140 ¹⁸⁶Re artificial 90 137 ¹⁸⁸Re natural 17 155

[0075] An adaptation of a calorimetric Re assay used in the field ofmetallurgy was used to determine the adsorption of the Re onto carrierparticles having a 70:30 iron:carbon ratio. In brief, a 0.1 to 0.5 mlsample was placed in a solution containing 1.0 ml of HCl, 1.3 ml of-furildoxime (6% in acetone), 0.5 ml of 10% stannous chloride andsufficient water to make 5.0 ml. The mixture was heated to 45° C. for 20minutes and allowed to cool to room temperature. The absorbance of Re inthe solution was measured at 532 nm. The sensitivity of the assay was toabout 5 mcg Re. These studies indicated that Re binding to a series ofcarbons varies from about 35% at 30 mg carbon in the adsorption mediumto about 90% at 180 mg carbon when incubated at ambient temperature. Aswith other drugs, the % binding of Re in the adsorption medium decreasesas the Re to carbon ratio increases. However, the binding of Re tocarbon does not correspond to the equilibrium binding isotherm ofLangmuir, and it is independent of temperature and pH. Release over 24hours of the Re into physiological saline at ambient temperature fromthe various carbons preloaded with adsorbed Re was 50% by weight.

[0076] Re was adsorbed onto carrier particles having iron:carbon ratiosof 70:30 and 85:15, respectively, by incubating the particles at ambienttemperature in an absorption medium containing buffered sodium chloride.Binding of the Re to the particles was determined by spectrophotometricassay. These studies showed that binding of Re to carrier particlesincreased with an increase in the ratio of carbon (i.e, in theparticles) to Re in the adsorption medium. The useful amount of adsorbedradioisotope will vary depending upon the particular results desired,for example, from 10 pgm to 700 ng. The proper amount should be easilydeterminable by any person having average skill in the art. The bindingparameters of for two different compositions of iron:carbon carrierparticles is shown in Table 2 below: TABLE 2 Iron:carbon ratio Amount ofRe (μg) in Binding of particles particles (mg) medium % Q (ng/mg) 85:15100 140 25.9 360 70:30 100 140 40.3 560

[0077] Less than 10% of the Re was released upon incubation under effluxconditions in saline over 24 hours. The low binding of Re to the carrierparticles is consistent with the low binding of other charged, smallionic molecules to activated carbon as compared with the high affinitybinding of hydrophobic aromatic molecules. These findings are consistentwith use of the carrier particles of the invention with adsorbed Re orTc as imaging and therapeutic agents.

EXAMPLE 4

[0078] Carrier particles having a 80:20 iron:carbon ratio were preparedas described above. Adsorption upon the particles of various types ofpharmaceutical agents at a range of concentrations of the pharmaceuticalagent in the absorption solution was performed to determine theabsorption curves and absorption constants for each compound as follows:

[0079] A. Antisense Oligonucleotide

[0080] A 16-mer anti-C-Myc oligonucleotide useful in antisensegene-directed therapy is an all phosphorothioate oligodeoxynucleotide,fluorescein-labeled at 5′ end (Macromolecular Resources, Fort Collins,Colo.). The oligo was dissolved in a stock adsorption solution made inTE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The concentration of theoligonucleotide in the buffer was determined assuming 1 AU₂₆₀=33 μg ofthe oligo, according to the manufacturer's recommendations. Unbound drugin adsorption supernatants was determined from the fluoresceinfluorescence (Exc. 495 nm, em. 549 nm) using a standard curve.

[0081] B. A Photosensitizer

[0082] Hematoporphyrin dihydrochloride (Sigma Chemical, USA, H-1875, Lot#23H0879) is a photosensitizer useful in tumor therapy. The compoundaccumulates by biological processes in certain types of tumor tissue.Upon exposure to light, such as provided by a laser, the compoundundergoes a chemical transformation to produce oxygen singlets that aretoxic to cells in which it has accumulated. A stock adsorption solutionwas prepared and drug concentration was determined by spectrophotometryaccording to G. Garbo et al. Anal. Biochem. 151:70-81, 1985, which isincorporated herein by reference in its entirety. (λ₄₀₃=327 mM⁻¹ in 1 NHCl). Unbound drug in adsorption supernatants was determined byspectrophotometry in an adsorption solution of 1 N HCl. The adsorptionequation determined by computer analysis using commercially availablesoftware was: C (μg/ml)=0.0984A²+1.85A at 403 nm.

[0083] C. An Anti-inflammatory Agent

[0084] 6-Mercaptopurine sodium salt (provided by Dr. Gruber, BurroughsWellcome, Lot #7P2774) is an anti-inflammatory agent. A stock adsorptionsolution was prepared by dissolving contents of a vial containing thecompound in 10 ml of MilliQ water. Drug concentration in adsorptionsupernatants was determined by spectrophotometry (standard curve: C(μg/ml)=9.0A −0.035 at 311 nm, R=0.9999, in 0.9% NaCl, pH adjusted to10.4 with NaOH).

[0085] D. An Anti-fungal Agent

[0086] Amphotericin B (Sigma Chemicals, A-4888, Lot 64H4005) is atherapeutically active agent useful against fungal infections. A stocksolution was prepared in 0.9% NaCl, 10 mM KOH at pH 12, withconcentration derived from drug weight corrected for the main compoundcontent (80%). Drug concentrations in adsorption supernatants determinedby spectrophotometry yielded the following equation for theconcentration curve: C (μg/ml)=3.61A²+18.1A+0.14 at 403 nm, R=0.9997, in0.9% NaCl, 10 mM KOH.

[0087] E. An Anti-cancer Agent

[0088] Camptothecin (Sigma Chemicals, C-9911, Lot #34H0956) is ananti-proliferative agent useful in treatment of certain types of tumor.A stock solution of 2 mg of camptothecin per ml was formed by dissolvingthe precise weight of the drug in the mixture of chloroform and ethanolat a ratio of 1:1 by volume (C/E 1:1). Drug concentration in adsorptionsupernatants as determined by spectrophotometry yield the followingequation for the concentration curve: C (μg/ml)=(16.7±0.26)A at 360 nmin C/E 1:1.

[0089] Camptothecin was also dissolved in DMSO and 0.9% saline solution,pH 3.0 at 1 mg/mL. Concentration was determined by absorbance at 253 nm(λ_(max)=253 nm in saline solution). Dilutions were made with 0.9%saline solution and MTC particles added to determine the Langmuirbinding isotherm.

[0090] The adsorption parameters determined are summarized in Table 3following: TABLE 3 Maximum Equilibra- adsorption Adsorption Iron:carbonAdsorption tion time (% of carrier constant Drug ratio medium (hrs)weight) (mg/ml)⁻¹ Oligonucleotide 80:20 TE buffer 1 1.48 ± 0.10  (1.0 ±2.1)10⁻² Type K Oligonucleotide 80:20 HEPES- 2 5.42 ± 0.34  (3.4 ±2.3)10⁻² Type K NS¹ Hematopor- 80:20 0.9% NaCl 1 5.97 ± 0.24  (3.0 ±1.8)10⁻⁴ phyrin Type K 6-MP 80:20 0.9% NaCl, 2 11.0 ± 0.97 0.24 ± 0.064Mercaptopurine Type K pH 10.4 Amphotericin B 80:20 0.9% NaCl 24  10.4 ±0.55  (1.1 ± 0.4)10⁻² Type K pH 12 (KOH) Camptothecin 80:20 C/E 1:1 3  0² — Type K Camptothecin 75:25 Saline   0.5 11 521 Type K solution

[0091] The results in Table 3 show that binding of the drug to thecarrier particles is highly influenced by the composition of theadsorption solution or medium. Camptothecin is a highly non-polarmolecule. In a highly non-polar adsorption medium (chloroforrn-ethanol),the drug does not preferentially leave the adsorption medium to adsorbto the carbon. However, in a more polar adsorption medium, it isbelieved that adsorption to the carrier particles would be entirelyacceptable. One of the factors that influences the adsorption of thedrug in the adsorption medium to the carbon in the carrier particle isthe hydrophobic Van der Waals interactions between the drug and theparticles. Alternatively, the drug can be dried onto the particles byevaporation techniques, for example, for adsorption of paclitaxel (PAC).

EXAMPLE 5

[0092] The carrier particles used for adsorption of paclitaxel (PAC)have an iron:carbon content of 70:30. The carbon is activated carbontype E. To analytically determine the iron content the followingprocedure was used. A portion of the sample was weighed (previouslydried in a vacuum desiccator) and washed at 2000° C., oxidizing allcarbon and iron present. During this procedure carbon was convertedquantitatively to CO₂ and volatilized, leaving a residue of Fe₂O₃. Theiron content was calculated by the formula. Fe═Fe₂O₃/1.42977, assumingno Fe₂O₃ was present initially. Carbon was assumed to be the remainingfraction. A second analysis of another portion of the sample wasperformed on a LECO carbon combustion analyzer. The sample was combustedand the CO₂ then measured, and total carbon was calculated. Iron andcarbon content calculated by both methods gave comparable results ofabout 69% by weight of elemental iron.

[0093] A. Binding Properties of Paclitaxel to Composite Particles

[0094] Drug adsorption was measured in two ways: 1) Initially a UVspectrophotometric assay was developed for screening drug bound to avariety of activated carbons. HPLC or spectrophotometric grade solventswere used throughout. The _(max) in ethanol was determined to be 220 nm.A Milton Roy Spectronic 21 spectrophotometer was used with 3 mL quartzcells. The wavelength of 254 nm was selected for UV analysis because itprovided good sensitivity for the drug. Little or no-contamination fromvarious assay techniques or materials was found at that wavelength. Thesame wavelength was used for the HPLC analysis. The UV assay was linearfor paclitaxel over the range 0.05-3.0 mg/mL.

[0095] In one test the carrier particles contained the KB-type carbon.It has a small pore size (˜40 nm effective radius), >1000 m²/gm surfaceareas, and good hardness. PAC adsorption capacity however was limited. Asurvey of some 20 other candidate activated carbons was reduced to threetypes with promising drug delivery properties, A, B, and E types ofcarbon. Iron powder alone was also tested. Each of these materials wasused at a concentration of 30 mg in citrated ethanol. The analysis by UVmethods gave the following binding results for 3 mg of PAC. Type Acarbon-74%, Type B carbon=65%, Type E carbon=33%, and iron powder=0% (nobinding) Types A and B carbon are both large pore, large surface area(1,800 m²/gm) carbons with drug release characteristics equivalent tothe E-type. E-type is a much harder carbon with a smaller surface areaand consequently better milling properties.

[0096] B. Paclitaxel Binding to Different Activated Carbons

[0097] Fractional binding (fb) (amount bound of initial amount of PAC)to activated carbon types A, B, and E increased with increasing amountof carbon (at fixed PAC concentration). Types A and B carbon could beshown to bind PAC 100% and to plateau in the binding curve at highactivated carbon content. Fractional bind of Type E was only 68%. Thebinding capacity, Q (expressed as % weight/weight drug carrier) wasshown to decrease with an increase in the amount of activated carbon.For type A carbon, the binding capacity, Q, increased from 8% to 44% fora decrease in carbon from 40 mg to 5 mg. The corresponding Q value forAC type E was about 5% to 7%.

[0098] Other studies of drug binding to type A carbon have suggestedthat a plateau in the fraction of drug bound as a function of the amountof absorber is a result of multilaminar drug coating on the surface ofthe carrier. In contrast, a linear increase in fraction bound isindicative of unilaminar coating, thus in keeping with the rules of theLangmuir isotherm analysis.

[0099] Our studies showed that Types A and E carbon have the ability toadsorb a considerable fraction (fb) of PAC in the adsorption medium andthat their binding capacity, Q, is also significant. On the other hand,carrier particles having a iron:carbon ratio of 70:30 (type E carbon)had both reduced capacity and fractional binding. These reduced valuesare in keeping with the proportionally lower carbon content of thecarrier particles as compared with carbon alone. In contrast, both thefb and Q values for the carrier particles with a higher binding capacitytype A carbon were less than 2%. This may be due to the inability of thepores in the carbon to withstand the compressive forces of the attritionmilling process during manufacture.

[0100] Despite the extensive binding of activated carbon Types A and Bto PAC, use of Type E carbon in carrier particles was preferred due tocommercial availability, and the proper balance between binding andrelease properties. In addition, Type E carbon is the preferredactivated carbon for use in a drug carrier because it has beenestablished to have U.S. Pharmacopoeia (22nd edition) quality. FIG. 6shows Langmuir adsorption plots for PAC binding to (—O—) carrierparticles with an iron:carbon ratio of 70%:30% Type E carbon and (— —)Type E carbon alone. Data were fit by simple unweighted linearregression.

[0101] Affinity (K_(m)) and maximal binding (Q_(m)) constants for PAC tothe carrier particles having an iron:carbon ratio of 70:30 (Type Ecarbon) were determined over a range of carrier amounts. Table 4 belowshows the results of adsorption isotherms of these compositions. Thevalues were determined graphically from FIG. 6 and Langmuir's equation.TABLE 4 Adsorber K_(m) (L/mg) Q_(m) (mg/mg %) Type E carbon alone 0.0149.1 Carrier particles (70:30) 0.014 3.6 with Type E carbon

[0102] PAC was loaded onto carrier particles and assayed by HPLC fordrug content, and then allowed to release drug for 24 hours, or longer.Measurements and fluid replacement took place in 2 hour intervals up to10 hours, and then daily thereafter. For the first 2 hours a magneticfield of 0.54 Tesla was applied to one set of two tubes containing theparticles, while no magnetic field was applied to the controls set oftubes. No statistical difference was found in the PAC release profilesbetween carrier particles on which the drug was magnetically retainedand those not subjected to a magnetic field (data not shown).

[0103] The PAC-adsorbed carrier particles and AC type E were pre-loadedusing varying amounts of PAC. After 72 hours at 37° C. in porcine sera(4 mL), free PAC was extracted one time with 5 mL of ethyl acetate. Theethyl acetate extracts were individually taken to dryness in air,reconstituted in 5 mL of methanol, and filtered through Millex GVfilters. Subsequent HPLC analysis indicated that cumulative drug releasein 24 hrs and after 72 hrs averaged 67% for the carrier particles (range53% to 86%) and averaged 64% for type E carbon. A low level of releasedPAC was subsequently validated independently by a bioassay system asdescribed below.

[0104] C. Magnetic Field Capture of Carrier Particles in a FlowingStream

[0105] A dynamic fluid flow circulation model similar to that describedby Senyei et al. (J. Appl. Phys. 49(6):3578, 1978) was used to evaluatethe forces and distances required to capture, retain, and accumulatecarrier particles or iron in a flowing fluid at low (water) and high(35% glycerol) viscosities. The glycerol was used to simulate bloodviscosity. Venous and arterial flow rates were simulated using flow rateand tubing diameter. Calibrated fluid flows were achieved with aprecision syringe pump. The magnet was a neodymium-iron-boron magnet(2.4×3.5 cm). The magnetic field was measured using a gaussmeter atvarious distances from the magnet surface. The magnet was placed 5 cmabove the effluent and moved in or out horizontally as needed to capturethe iron or carrier particles. Complete (100%) capture, or retention,was the end-point of the experiment. Carrier particles or iron powderwas introduced as a suspension through a syringe valve approximately 40cm from the pump injection syringe and 40 cm from the effluent. Inglycerol, the magnetic field necessary to retain 100% of iron or of thePAC-adsorbed particles was about 10% greater than in water.

[0106] A bioassay of tumor cytotoxicity from carrier particles loadedwith PAC was carried out using a human squamous carcinoma cell line,SCC-9. Cell viability was independently determined following six days ofincubation with each of: I) paclitaxel, 2) PAC-loaded carrier particles,3) type E activated carbon, 4) free carrier particles, and 5) elementaliron. The assay was a slight variation of Mosmann's MTT cytotoxicitytechniques. This spectrophotometric assay measures the quantitativereduction of the yellow tetrazolium salt of3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl-tetrazolium bromide] to itspurple formazan derivative by the mitochondria of living cells. Theamount of carrier used in these experiments was 0.5-117 g/mL. None ofthe controls, including DMSO (used for paclitaxel alone) were cytotoxicto the SCC-9 tumor cells.

[0107] A separate long-term CF clonogenic assay was evaluatedconcurrently. In this assay, cells were plated on 35 mm petri dishes andexposed to drug and controls as outlined above. Treated cells wereincubated for 2-3 weeks to allow time for colonies to establish. Thesecolonies were fixed in 2% acetic acid and 8% ethanol and stained withcrystal violet. The colonies were counted under a Bella Glass PlateReader. The IC₅₀ for the CF assay was 1×10⁻² g/mL forpaclitaxel-adsorbed micro carriers, and 3×10⁻³ g/mL for paclitaxelalone. The IC₅₀ by MTT assay for PAC released from carrier particles andpaclitaxel alone were identical at both 500 and 1000 plated cells,9×10⁻³ g/well. The MTT assay for drug or chemically induced cytotoxicityis a surrogate marker for “true” cell kill. Therefore, usually more drugis required to demonstrate a given level of cell kill with the colonyforming (CF) assay. Consequently, the dose response curve is shifted toa higher concentration level of paclitaxel.

[0108] Furthermore, no adverse effects were found from the magneticfield retention of drug-free carrier particles on the tumor cells inculture. The IC₅₀ for free paclitaxel alone was identical (5 ng/mL) topaclitaxel released from the carrier particles with or without magnet (5ng/mL). In both instances the carrier particles showed no adverseeffects on the cells during the in vitro cytotoxicity evaluation.

[0109] These results demonstrated that pharmacologically activepaclitaxel can be released from the carrier particles of the invention,and that the chemical analysis of adsorbed and released drug can beconfirmed biologically. Similar dose-response curves were obtained forfree paclitaxel and paclitaxel desorbed from the carrier particles.

[0110] Iron:carbon carrier particles having various iron:carbon ratiosin the range from 95:5 to 45:55 were prepared as described hereinutilizing type A activated carbon as the carbon source. The particleswere incubated in an adsorption solution containing 0.67 mg/ml ofdoxorubicin (Dox) in saline-citrate buffer (pH 7.4) to determine thecapacity and binding of the Dox to composite particles of differentcomposition. Table 5 below shows the results of these studies. TABLE 5Iron:Carbon 95:5 85:15 75:25 65:35  60:40  55:45 45:55 ratio *Binding10.9% 12.2% 14.1% 15.6% 15.9% 15.8% 15.7% Capacity ** % Binding 69.0%78.7% 89.4 99.5%  100%  100% 99.7% Tap Density 1.39 0.90 0.46 0.48 0.490.48 0.62 (gm/cm³) Average Size 0.73 0.80 0.74 0.74 0.76 0.71 0.82 (μ)

[0111] The data in Table 5 shows the relationship between theiron:carbon content of the particles and drug binding to the particles.

EXAMPLE 6

[0112] Additional studies were conducted to compare the effect of thecomposition of the adsorption solution on the binding of the Dox tocarrier particles having an iron:carbon ratio in the range 60:40 to80:20. The following six test adsorption media compositions wereformulated to provide sufficient viscosity to keep the carrier particlesphysically separate during adsorption of the Dox. The particles werefirst placed in the viscosity agent and the Dox/saline solution waslater added.

[0113] 1. 10% mannitol; 2% sodium carboxymethyl cellulose (CMC) (mediumviscosity); 2% polyvinyl pyrrolidone (PVP in 50 mM citrate phosphatebuffer

[0114] 2. 5% mannitol; 2% CMC; 2% PVP in 50 mM citrate phosphate buffer.

[0115] 3. 5% mannitol; 2% CMC; 2% PVP; 5% sorbitol in 50 mM citratephosphate buffer.

[0116] 4. 10% mannitol; 1% CMC; 2% PVP (K15) in 10 mM potassiumphosphate buffer (pH 7.4).

[0117] 5. 10% mannitol; 1% sodium CMC; potassium phosphate buffer (pH7.4).

[0118] 6. 5% sorbitol; 1% sodium CMC; 2% PVP (K15); 5% mannitol; inpotassium phosphate buffer (pH 7.4)

[0119] Adsorption studies using each of the above adsorption mediashowed that the highest adsorption of Dox to the carrier particles wasobtained using formulae 4, 5, and 6 of the above group.

[0120] Alternatively, the particles may be combined with the Dox/salinesolution first and the viscosity agent added later. In this process, 10%mannitol and 5% CMC provided desirable results.

EXAMPLE 7

[0121] Certain porphyrins are photosensitizing compounds useful inphotodynamic therapy against tumors. The so called “second generation”photosensitizers possess major adsorption peaks at wavelengths 650 nmand many of these compounds are in clinical trials in the U.S., Japan,and Europe. Several classes of photosensitizers were screened forcomparative binding to iron:carbon particles of various composition. Thewavelength near the activation wavelength (often the max) of aparticular photosensitizer was used for quantitative drug measurements.It was found that concentrations of various porphyrins at 80 mcg/ml(0.11 mM) in phosphate buffered saline (PBS) pH 7.4 were convenient forinitial binding studies. The photosensitizers tested werehematoporphyrin derivative (HPD); benzoporphyrin derivative monoacid A(BPD-ma); Photofrin® porfimer sodium (PF2); and clorin e6. For thebinding studies, 10 mg of carbon or 50 mg of iron:carbon particles wereoptimum. An octanol/buffer (pH 7.4) partition coefficient for the fourcompounds was as follows: HPD=1; chlorin e6=1.1; PF2=0.1; andBPD-ma=4000.

[0122] The results of the binding studies are summarized in Table 6below TABLE 6 HPD Clorin e6 PF2 BPD-ma iron:carbon % binding % binding %binding % binding ratio mg/mg % % mg/mg % % mg/mg % % mg/mg % % 30:700.4 37.0 0.7 68.8 0.08  7.2 0.25 23.0 Type E 30:70 0.5 41.9 0.8 69.90.13 11.4 0.33 30.1 Type A

[0123] In order to achieve higher loading levels of BPD-ma, the bindingcapacity and fractional binding of the drug to four prototypeiron:carbon carrier particles (MTCs, or, magnetic targetted compounds)was tested using 1.4 mM drug in isopropanol (with 0.5% 0.02 M aceticacid) as the adsorption medium and a longer equilibration period of 18hours. As shown in Table 7 below, by this technique, a 30-fold increasein binding capacity from a 10-fold increase in the initial concentrationof the drug was obtained. TABLE 7 SUMMARY OF BINDING AND RELEASE OFBPD-ma A A E E Carbon Type MTC26.2 MTC15.1 MTC5241 MTC5273 iron:carbonratio 70:30 60:40 70:30 60:40 Binding capacity  9.5 13.9 11.0 11.7(mg/mg %) fractional binding % 43.5 63.5 53.6 57.7 % release 54.7 13.7 9.1  7.9 (mg/mg bound)

[0124] These studies showed that the carrier particles using Type Acarbon in a 60:40 iron:carbon ratio (MTC 15.1) were significantlydifferent than the other particles tested with respect to bindingcapacity and fractional binding of the total amount of the drug used.When a magnetic field was used facilitate washing each of the carriersfree from unbound BPD-ma, the MTC 15.1 carrier particles did not give aclear solution as others did. It was assumed that a significant amountof carbon was released from the surface of the particles in the processof binding. By contrast, the carrier particles using TypeA carbon in a70:30 iron:carbon ratio (MTC 26.2) gave up bound BPD-ma more efficientlythan the other carriers tested while retaining a good level of initialadsorption.

EXAMPLE 8

[0125] In one sterilization technique, particles may be rendered sterilein a glass vial using gamma irradiation. In this system, at least 1000vials may be sterilized at a time, using 2.5 to 3.5 Mrad's of gammairradiation from a cobalt source. For instance, both particle lots 0198(made with carbon A) and 0498 (made with carbon KB) were renderedsterile in this fashion. Each was tested after sterilization and foundto have retained all manufactured properties, such as particle sizedistribution and doxorubicin binding capacity. Similarly, the aqueoussolution of excipients may be rendered sterile through autoclavetreatment at 121° C. for thirty minutes. For example, lot 0398 (100mL/vial) and lot 0598 (20 mL/vial) were sterilized in this fashion. Eachwas tested after sterilization and found to have retained allmanufactured properties, such as ability to suspend particles in avehicle suitable for human administration

EXAMPLE 9

[0126] Camptothecin has been shown to bind to MTC particles preparedwith K carbon in a weight ratio of 75:25 Fe:C. Camptothecin may be boundto particles out of 0.9% saline solution or 10% lactose solution, but ispreferentially bound from saline. The following graph shows bindingacross a range of solution concentrations, and indicate a maximumbinding capacity of approximately 110 μg/mg MTC (11%) from saline.

[0127] Camptothecin has two commercial derivatives, topotecan andirinotecan, and a third chemical derivative in clinical trials calledaminocamptothecin. These derivatives represent minor chemical changes tothe camptothecin molecule. These and other chemical derivatives shouldalso bind.

EXAMPLE 10

[0128] Methotrexate has been shown to bind to MTC particles preparedwith K carbon in a weight ratio of 75:25 Fe:C. Methotrexate may be boundto particles out of 0.9% saline solution or 10% lactose solution, but ispreferentially bound from saline. The following graph shows bindingacross a range of solution concentrations, and indicate a maximumbinding capacity of approximately 100 μg/mg MTC (10%) from saline.

[0129] Methotrexate has one chemical derivative, aminopterin. Thisderivative represents a minor chemical change to the methotrexatemolecule. These and other chemical derivatives should also bind.Methotrexate belongs to a class of molecules called folate antagonists.These molecules interfere with the synthesis of folate in a cancer cell.Folate antagonists are structurally similar, as their mode of actionrequires that they bind and inhibit the action of a specific enzyme.Examples of other folate antagonists are pyritrexin; 10-ethyl,10-deaza-aminopterin; trimetrexate; 5,10-deaza,10-proparglyfolic acid;and 5,10-dideazatetrahydrofolate. These and other folate antagonistsshould also bind.

EXAMPLE 11

[0130] Paclitaxel has been shown to bind to MTC particles prepared withK carbon in a weight ratio of 75:25 Fe:C. Paclitaxel may be bound toparticles out of ethanol or cremaphor EL formulation, but ispreferentially bound from a citrate buffered aqueous ethanol mixture.The following graph shows binding across a range of solutionconcentrations, and indicate a maximum binding capacity of approximately160 μg/mg MTC (16%) from citrated buffered aqueous ethanol. The bindingextends to all concentrations between 0% and 16%.

[0131] Paclitaxel has been bound to particles composed of three othercarbons. The maximum binding observed in each is shown in the followingtable: TABLE 11 Carbon Maximum binding type (μg/mg) A 190 B 174 E  82 KB160

[0132] Paclitaxel is a chemical derivative of taxol, which has anotherchemical derivative, taxotere. There are other taxol derivatives, mostsemi-synthetic, that have similar structures to taxol and paclitaxel.These derivatives represents minor chemical changes to the taxolmolecule. These and other chemical derivatives should also bind.

EXAMPLE 12

[0133] Verapanil has been shown to bind to MTC particles prepared with Kcarbon in a weight ratio of 75:25 Fe:C. Verapamil may be bound toparticles out of lactose or saline solution, but is preferentially boundfrom an aqueous saline solution. The following graph shows bindingacross a range of solution concentrations, and indicate a maximumbinding capacity of approximately 140 μg/mg MTC (14%) from saline. Thebinding extends to all concentrations between 0% and 14%.

EXAMPLE 13

[0134] Ferrocarbon particles were prepared and doxorubicin was adsorbedfor a resulting dose solution of 0.4 mg/ml doxorubicin and 5.0 mg/mlcarrier. Selective catheterization of the hepatic artery was performedfor delivery to Yorkshire domestic swine. Animals received 3-6 pulsedinfusions every 10-30 minutes for a cumulative dose of 14.2-18 mgdoxorubicin. An external magnet was held in position during the infusionprocedure and for 15-30 minutes directly thereafter. Animals wereevaluated over 28 days and then sacrificed. Histopathological evaluationshowed that 18 mg doxorubicin given in 7.5 mL infusion cycles every 15minutes was the maximum tolerated dose. This determination was basedprimarily on the occurrence of hepatic necrosis and portal area changes.

What is claimed is:
 1. A magnetically responsive composition comprisingparticles including carbon and iron, wherein the carbon is substantiallyuniformly distributed throughout the particle volume, wherein thecross-sectional size of each particle is less than about 5 μm, andwherein the carbon is selected from the group consisting of types A, B,E, K, KB, and chemically modified versions thereof.
 2. The compositionof claim 1, wherein the particles are about 0.1 μm to 5.0 μm incross-sectional size, each particle including a weight ratio of iron tocarbon in the range from about 95:5 to 50:50, and having a therapeuticamount of doxorubicin adsorbed thereon.
 3. The composition of claim 2,wherein the weight ratio of iron to carbon is from about 80:20 to 60:40.4. The composition of claim 3, wherein the average amount of doxorubicinis up to 20% of the mass of the particle.
 5. The composition of claim 1,wherein the particles are about 0.1 μm to 5.0 μm in cross-sectionalsize, each particle including a weight ratio of iron to carbon in therange from about 95:5 to 50:50, and having a therapeutic amount ofcamptothecin, or an analog thereof, adsorbed thereon.
 6. The compositionof claim 5, wherein the weight ratio of iron to carbon is from 80:20 to60:40.
 7. The composition of claim 6, wherein the average amount ofcamptothecin is up to 20% of the mass of the particle.
 8. Thecomposition of claim 5, wherein the analog of camptothecin is topotecan.9. The composition of claim 8, wherein the weight ratio of iron tocarbon is from 80:20 to 60:40.
 10. The composition of claim 9, whereinthe average, amount of topotecan is up to 20% of the mass of theparticle.
 11. The composition of claim 5, wherein the analog ofcamptothecin is irinotecan.
 12. The composition of claim 11, wherein theweight ratio of iron to carbon is from 80:20 to 60:40.
 13. Thecomposition of claim 12, wherein the average amount of irinotecan is upto 20% of the mass of the particle.
 14. The composition of claim 5,wherein the analog of camptothecin is aminocamptothecin.
 15. Thecomposition of claim 14, wherein the weight ratio of iron to carbon isfrom 80:20 to 60:40.
 16. The composition of claim 15, wherein theaverage amount of aminocamptothecin is up to 20% of the mass of theparticle.
 17. The composition of claim 1, wherein the particles areabout 0.1 μm to 5.0 μm in cross-sectional size, each particle includinga weight ratio of iron to carbon in the range from about 95:5 to 50:50,and having a therapeutic amount of taxol, or an analog thereof, adsorbedthereon.
 18. The composition of claim 17, wherein the taxol analog istaxotere.
 19. The composition of claim 18, wherein the weight ratio ofiron to carbon is from 80:20 to 60:40.
 20. The composition of claim 19,wherein the average amount of taxotere is up to 20% of the mass of theparticle.
 21. The composition of claim 17, wherein the taxol analog ispaclitaxel.
 22. The composition of claim 21, wherein the weight ratio ofiron to carbon is from 80:20 to 60:40.
 23. The composition of claim 22,wherein the average amount of paclitaxel is up to 20% of the mass of theparticle.
 24. The composition of claim 1, wherein the particles areabout 0.1 μm to 5.0 μm in cross-sectional size, each particle includinga weight ratio of iron to carbon in the range from about 95:5 to 50:50,and having a therapeutic amount of verapamil, or an analog thereof,adsorbed thereon.
 25. The composition of claim 24, wherein the weightratio of iron to carbon is from 80:20 to 60:40.
 26. The composition ofclaim 25, wherein the average amount of verapamil is up to 20% of themass of the particle.
 27. The composition of claim 1, wherein theparticles are about 0.1 μm to 5.0 μm in cross-sectional size, eachparticle including a weight ratio of iron to carbon in the range fromabout 95:5 to 50:50, and having a therapeutic amount of a folateantagonist adsorbed thereon.
 28. The composition of claim 27, whereinthe folate antagonist is methotrexate.
 29. The composition of claim 28,wherein the weight ratio of iron to carbon is from 80:20 to 60:40. 30.The composition of claim 29, wherein the average amount of methotrexateis up to 20% of the mass of the particle.
 31. The composition of claim27, wherein the folate antagonist is aminopterin.
 32. The composition ofclaim 31, wherein the weight ratio of iron to carbon is from 80:20 to60:40.
 33. The composition of claim 32, wherein the average amount ofaminopterin is up to 20% of the mass of the particle.
 34. Thecomposition of claim 27, wherein the folate antagonist is pyritrexin.35. The composition of claim 34, wherein the weight ratio of iron tocarbon is from 80:20 to 60:40.
 36. The composition of claim 35, whereinthe average amount of pyritrexin is up to 20% of the mass of theparticle.
 37. The composition of claim 27, wherein the folate antagonistis 10-ethyl, 10-deaza-aminopterin.
 38. The composition of claim 37,wherein the weight ratio of iron to carbon is from 80:20 to 60:40. 39.The composition of claim 38, wherein the average amount of 10-ethyl,10-deaza-aminopterin is up to 20% of the mass of the particle.
 40. Thecomposition of claim 27, wherein the folate antagonist is trimetrexate.41. The composition of claim 40, wherein the weight ratio of iron tocarbon is from 80:20 to 60:40.
 42. The composition of claim 41, whereinthe average amount of trimetrexate is up to 20% of the mass of theparticle.
 43. The composition of claim 27, wherein the folate antagonistis 5,10-deaza, 10-proparglyfolic acid.
 44. The composition of claim 43,wherein the weight ratio of iron to carbon is from 80:20 to 60:40. 45.The composition of claim 44, wherein the average amount of 5,10-deaza,10-proparglyfolic acid is up to 20% of the mass of the particle.
 46. Thecomposition of claim 27, wherein the folate antagonist is5,10-dideazatetrahydrofolate.
 47. The composition of claim 46, whereinthe weight ratio of iron to carbon is from 80:20 to 60:40.
 48. Thecomposition of claim 47, wherein the average amount of5,10-dideazatetrahydrofolate is up to 20% of the mass of the particle.49. The composition of claim 1, wherein the particles are about 0.1 μmto 5.0 μm in cross-sectional size, each particle including a weightratio of iron to carbon in the range from about 95:5 to 50:50, andhaving a therapeutic amount of a radioisotope adsorbed thereon.
 50. Thecomposition of claim 49, wherein the amount of radioisotope is fromabout 10 pgm to 700 ng.
 51. The composition of claim 1, wherein theparticles are about 0.1 μm to 5.0 μm in cross-sectional size, eachparticle including a weight ratio of iron to carbon in the range fromabout 95:5 to 50:50, and having a diagnostic amount of a radioisotopeadsorbed thereon.
 52. The composition of claim 51, wherein the amount ofradioisotope is from about 10 pgm to 700 ng.
 53. The composition ofclaim 1, wherein the particles are about 0.1 μm to 5.0 μm incross-sectional size, each particle including a weight ratio of iron tocarbon in the range from about 95:5 to 50:50, and having a therapeuticamount of a biologically active substance adsorbed thereon.
 54. Thecomposition of claim 53, wherein the biologically active substance is adrug, a radioactive substance, or genetic material.
 55. The compositionof claim 54, wherein the radioactive substance is ¹⁸⁶Re, ¹⁸⁸Re, ¹²³I,¹²⁵I, or ⁹⁰Y.
 56. The composition of claim 1, wherein the particles areabout 0.1 μm to 5.0 μm in cross-sectional size, each particle includinga weight ratio of iron to carbon in the range from about 95:5 to 50:50,and having a diagnostic amount of a biologically active substanceadsorbed thereon.
 57. The composition of claim 56, wherein thebiologically active substance is a radioisotope, a contrast agent, a dyeor genetic material.
 58. The composition of claim 57, wherein theradioactive substance is ¹⁸⁶Re, ¹⁸⁸Re, or ⁹⁹Tc.
 59. A kit foradministering a biologically active substance to an in vivo site in apatient comprising a receptacle containing: a) unit dose of dryferrocarbon particles between about 0.1 μm and 5.0 μm in cross-sectionalsize, each particle including a ratio of iron to carbon in the range ofabout 95:5 to 50:50 and having the carbon distributed throughout thevolume of the particle; and b) one or more dry excipients in an amountthat enhances adsorption of a biologically active substance to theparticles when in an aqueous solution.
 60. The kit of claim 59, whereinthe unit dose is from about 0.05 to about 0.5 grams of the particles.61. The kit of claim 59, wherein the excipients include a biologicallycompatible polymer for separating the particles when added to theaqueous solution.
 62. The kit of claim 59, wherein the excipientsinclude mannitol, sodium carboxy methyl cellulose, or combinationsthereof.
 63. The kit of claim 59, wherein the contents of the kit arecombined with a commercially prepared formulation of a biologicallyactive substance.
 64. A kit for administering a biologically activesubstance to an in vivo site in a patient comprising: a) A firstreceptacle comprising a unit dose of ferrocarbon particles between about0.1 μm and 5 μm in cross-sectional size, each particle including a ratioof iron to carbon in the range from about 95:5 to 50:50 with the carbondistributed throughout the volume of the particle; and b) A secondreceptacle comprising an aqueous solution comprising one or moreexcipients in an amount that enhances adsorption of a biologicallyactive substance to the particles when in an aqueous solution.
 65. Thekit of claim 64, wherein the unit dose is from about 0.05 to about 0.5grams of the particles.
 66. The kit of claim 64, wherein the excipientsinclude a biologically compatible polymer for separating the particleswhen added to the aqueous solution.
 67. The kit of claim 64, wherein theexcipients include mannitol, sodium carboxy methyl cellulose, orcombinations thereof.
 68. The kit of claim 67, wherein the amount ofmannitol is 10% and the amount of carboxy methyl cellulose is 5%. 69.The kit of claim 64, wherein the contents of the kit are combined with acommercially prepared formulation of a biologically active substance.70. The kit of claim 64, wherein the unit dose of ferrocarbon particleshas been sterilized by means of gamma irradiation.
 71. The kit of claim64, wherein the aqueous solution comprising the excipients has beensterilized by means of autoclave.
 72. A method of sterilizing acomposition comprising ferrocarbon particles comprising the use of gammairradiation.
 73. The method of claim 71, wherein the amount of gammairradiation used is from 2.5 to 3.5 Mrads.
 74. A kit for administering abiologically active substance to an in vivo site in a patient comprisinga unit dose of ferrocarbon particles between about 0.1 μm and 5 μm incross-sectional size, each particle including a ratio of iron to carbonin the range from about 95:5 to 50:50 with the carbon distributedthroughout the volume of the particle.